Mutation of a stromal C-terminal threonine residue of Photosystem II subunit S slows down NPQ induction and speeds up relaxation.
In order to prevent damage by excess light, light harvesting antennae can switch to an energy dissipative mode (termed non-photochemical quenching, NPQ). In higher plants, this switch is facilitated by the presence of Photosystem II subunit S (PsbS) protein, which was discovered 25 years ago. While the role of PsbS in induction of NPQ was soon found to require protonation of key glutamate residues facing the thylakoid lumen, a complete understanding of how NPQ is subsequently initiated is still lacking. Recent work on Norway spruce suggests that reversible phosphorylation at a few conserved residues of PsbS may affect its role in regulation of NPQ. Here we assessed PsbS phosphorylation changes in Arabidopsis thaliana plants, but these remained undetectable under control and combined chilling and high light stress conditions. We therefore used a genetic approach to assess potential functional implications of phosphorylation at threonine-259 (T259). Functional evaluation of point mutations at T259 in the background of PsbS knock-out mutant npq4 showed that neither phosphomimetic, phosphosubstitution, nor phosphonull substitutions could rescue NPQ activity to the level of the unperturbed protein, inconsistent with regulation via reversible phosphorylation. Instead, all residue substitutions at T259 gave rise to significantly impaired induction and accelerated NPQ recovery, while protein accumulation and thylakoid membrane localisation were not affected. We suggest that these results point to a role for the C-terminus in the propensity or stability of hydrophobic interactions between PsbS and LHCII antenna proteins to initiate the quenched state.